Helicases are motor proteins that use the free energy from NTP hydrolysis to translocate along nucleic acids and to separate the strands of dsDNA or RNA, catalyze branch migration, or remove proteins bound to nucleic acids. These functions of helicases are required in most DNA and RNA metabolic processes; therefore, helicases are ubiquitous and certain mutations in helicases lead to genetic instabilities and diseases such as many forms of cancer and premature aging in humans. In the previous grant period, we made a comprehensive effort to understand the enzymatic mechanisms of three different helicases: ring-shaped T7 helicase and Rho [unreadable] proteins, and superfamily II hepatitis C virus helicase. Many of the proposed studies will test the hypotheses generated from the last grant period. Kinetic analysis of two different ring-shaped helicases aims to achieve both a detailed and a general understanding of the workings of hexameric helicases. The hexamer has six potential NTPase sites that can hydrolyze NTP with various sequences. We have dissected the NTPase reaction into several steps and determined the rate limiting step. In specific Aim 1, we will investigate cooperativity amongst the subunits of hexameric T7 helicase and E. coli Rho in NTP hydrolysis by systematically characterizing the inhibition of NTPase by the non-hydrolyzable nucleotide analogs. We will also study the activities of crosslinked and defined mixed hexamers of T7 helicase containing one or more functionally defective mutant subunit. Measurement of the ssDNA translocation rate and the dsDNA unwinding rate has shown that the movement of T7 helicase is restricted by duplex DNA. In Aim 2, we will characterize the unwinding reaction and the coupling of NTPase to strand separation to determine if the helicase's activity is tightly coupled to NTP hydrolysis or if the helicase undergoes e.g., idling or backward movement at the unwinding junction, thereby requiring additional NTP. In Aim 3, we will study the cooperative actions of the T7 helicase-T7 DNA polymerase complex at the replication fork. We will investigate the mechanism by which T7 DNA polymerase activates the helicase activity and investigate the fidelity of DNA synthesis by the helicase-polymerase complex. [unreadable] [unreadable]